To understand the relationship between hypertension and insulin resistance we are studying insulin signal transduction pathways related to nitric oxide (NO) production in vascular endothelium. Human forearm blood flow studies suggest that physiological concentrations of insulin cause vasodilation of small vessels via the production of NO endothelial cells. Furthermore, the degree of insulin sensitivity exhibited for this response is positively correlated with insulin sensitivity for glucose uptake. Thus, insulin resistance may contribute to the pathogenesis of hypertension under some conditions. We used an NO electrode to directly measure NO at nanomolar concentrations to characterize the insulin response of human umbilical vein endothelial cells (HUVEC) in primary culture. In addition, we developed a novel method for transiently transfecting endothelial cells in primary culture and selecting the transfected cells using a fluorescently activated cell sorter. By overexpressing wild-type, constitutively active, or dominant inhibitory forms of various signaling molecules, we have identified the insulin receptor tyrosine kinase, PI 3-kinase, and Akt as necessary components of insulin signaling pathways related to production of NO. Moreover, we have ruled out an important role for Ras. We have also used the NO-specific fluorescent dye DAF-2 to visualize production of NO in single cells and dissect the mechanisms whereby insulin regulates activity of endothelial nitric oxide synthase. We found that insulin stimulates activation of eNOS by a clacium independent mechanism involving phosphorylation of eNOS by Akt.